Liposomes are one of a number of lipid-based materials used as biological carriers and have been used effectively as carriers in a number of pharmaceutical and other biological situations, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcriptional factors and other cellular vectors into a variety of cultured cell lines and animals. Successful clinical trials have examined the effectiveness of liposome-mediated drug delivery for targeting liposome-entrapped drugs to specific tissues and specific cell types. See, for example, U.S. Pat. No. 5,264,618, which describes a number of techniques for using lipid carriers, including the preparation of liposomes and pharmaceutical compositions and the use of such compositions in clinical situations. However, while the basic methodology for using liposome-mediated vectors is well developed, improvements in the materials used in the methods, both in terms of biocompatability and in terms of effectiveness of the carrier process, are still desirable.
In particular, the expression of exogenous genes in humans and/or various commercially important animals will ultimately permit the prevention and/or cure of many important human diseases and the development of animals with commercially important characteristics. Genes are high molecular weight, polyanionic molecules for which carrier-mediated delivery usually is required for DNA transfection of cells either in vitro or in vivo. Therefore it is of interest to develop lipid transfection vectors which will enhance both the delivery and the ultimate expression of the cloned gene in a tissue or cell of interest. Since in some instances a treatment regimen will involve repeated administration of a gene (or other pharmaceutical product), it also is of interest that the lipid carriers be nontoxic to the host, even after repeated administration.
Relevant literature
Amphiphilic phosphatidylethanolamine conjugates for functionalization of liposomes are disclosed in Law et al., (1986) Tetrahedron Letters, 27:271-274. A method for synthesis of 1,2-dipalmitoyl-SN-glycero-3-phosphoester is disclosed in Bruzik et al., (1986) J. Org. Chem., 51:2368-23270. The o-methyl ester of dipalmitoyl phosphatidylcholine was prepare as an intermediate in this synthesis.
Use of liposomes as carriers for DNA is described in a number of publications, including the following: Friedmann, (1989) Science, 244:1275-1281; Brigham et al., (1989) Am. J. Med. Sci., 298:278-281; Nabel et aL, (1990) Science, 249:1285-1288; Hazinski et al., (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang, (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855. Other publications relating to liposomes describe liposomes coupled to ligand-specific, cation-based transport systems (Wu and Wu, (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA expression vectors (Nabel et al., (1990) Science, 249: 1285-1288); Wolff et al., (1990) Science, 247:1465-1468).
The Brigham et al. group Am. J. Med. Sci. (1989) 298:278-281 and Clinical Research (1991) 39 (abstract)! have reported in vivo transfection of lungs of mice following either intravenous or intratracheal administration of a DNA liposome complex. See also Stribling et al., (1992) Proc. Nat'l Acad. Sci. USA 89:11277-11281, which reports the use of liposomes as carriers for aerosol delivery of transgenes to the lungs of mice, and Yoshimura et aL (1992) Nucleic Acids Research 20:3233-3240.
Cationic lipid carriers have been reported to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Nat'l. Acad. Sci. USA (1987) 84:7413-7417); mRNA (Malone and Keloff, Proc. Nat'l. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265: 10189-10192) in functional form.